While the expression ofimmunoglobulins and T cell receptors on cancer cells has been well-established for decades, the potential roles and mechanisms of action of these cancerous antigen receptors have not been fully elucidated. A monoclonal antibody designated as RP215, which reacts specifically with the carbohydrate-associated epitope located on the heavy chain region of cancerous immunoglobulins and T cell receptors, was used as a unique probe to study the rolesof antigen receptors in the immunologyofcancer cells. Through extensive cell-based biological and immunological studies, it was found that both anti-antigen receptors and RP215 demonstrated similar actions on the gene regulations involved in the growth/proliferation ofcancer cells, as well as on toll-like receptors involved in innate immunity. In addition, RP215-specific cancerous immunoglobulins are believed to capture or neutralize circulating antigen/antibodies which may be harmful to cancer cells within the human body. In contrast to normal B and T cells and their respective receptors in the conventional immune system, cancer cells co- express both immunoglobulins and T cell receptors and immune protection is exercised by unique mechanisms. For example, these cancercell-expressed antigen receptors display a lack of class switching, limited hyper-mutation, aberrant glycosylations and a strong influence on the toll-like receptors ofcancer cells. Therefore, it is hypothesized that both normal and cancerous immune systems may co-exist and operate simultaneously within the human body. The balance of these two immune factors for respective surveillance and protection may be relevant to the outcome ofcancer immunotherapy in humans. A potential therapeutic strategy is being developed by using RP215 as a drug candidate to target cancer cells based on these observations.
As highlighted in the above sections, the classical or alternative NF-κB pathways appear to be the main mediators of most cellular events stemming from LTβR signaling that contribute to its pro- and anti-oncogenic effects. However, a number of reports suggest that this is not always the case. In fact, some anti-oncogenic effects of LTβR signaling leading to cancercell death were reported to be mediated by other downstream components such as the reactive oxygen species-induced apoptosis signal-regulating kinase (ASK1)  and caspases (e.g., caspase 3 and 8) [66,71,73]. On the other hand, only few pro-tumorigenic effects of LTβR signaling were found to result from activation of mediators other than NF-κB. Ammirante et al. (2010) reported that LTβR activation in prostate carcinoma cells by lymphotoxin expressed on B cells infiltrating regressing tumors after castration was required for IKKα translocation to the nucleus and STAT3 activation, nevertheless a collaboration with another unidentified critical cytokine/receptor activating STAT3 was predicted . Although JNK has been shown to be activated by LTβR (Figure 2) and to be implicated incancer, in promoting or suppressing it , no report has so far addressed whether this kinase is involved incancer-related LTβR activity.
Clinical observations and mouse models have suggested that inflammation can be pro-tumorigenic. Since chemokines are critical in leukocyte trafficking, we hypothesized that chemokines play essential rolesin inflammation-associated cancers. Screening for 37 chemokines in prostate cancercell lines and xenografts revealed CXCL16, the ligand for the receptor CXCR6, as the most consistently expressed chemokine. Immunohistochemistry and/or immunofluorescence and confocal imaging of 121 human prostate specimens showed that CXCL16 and CXCR6 were co-expressed, both on prostate cancer cells and adjacent T cells. Expression levels of CXCL16 and CXCR6 on cancer cells correlated with poor prognostic features including high-stage and high-grade, and expression also correlated with post-inflammatory changes in the cancer stroma as revealed by loss of alpha-smooth muscle actin. Moreover, CXCL16 enhanced the growth of CXCR6-expressing cancer and primary CD4 T cells. We studied expression of CXCL16 in an additional 461 specimens covering 12 tumor types, and found that CXCL16 was expressedin multiple human cancers associated with inflammation. Our study is the first to describe the expression of CXCL16/CXCR6 on both cancer cells and adjacent T cells in humans, and to demonstrate correlations between CXCL16 and CXCR6 vs. poor both prognostic features and reactive changes incancer stoma. Taken together, our data suggest that CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancercell growth and by inducing the migration and proliferation of tumor- associated leukocytes.
Analytics, Landsberg, Germany), with Solvent degasser PN7520 autosampler (PN5300 model, Postnova Analytics) and Isocratic LC–Pump PN1130 (Postnova Analytics, Landsberg). The channel had a 0.0350 cm thick spacer and was 27.5 cm long (tip to tip) and 2 cm wide. A regen- erated cellulose membrane having a nominal cut-off of 10 kDa was fixed over a porous frit wall. Samples diluted in water (1:200) were injected (10–50 μL) and eluted with particle- free Milli-Q water under a channel tip flow of 4.5–0.5 mL/min, for a duration of 35 min. The focusing step consisted of a flow delivered by the injection port of 4.2 mL/min for 2 min. The cross- flow gradient was 4–0 mL/min (35 min). After 35 min a tip flow of 0.5 mL/min was maintained for 20 min to clean up the channel. The system was coupled with a variable wavelength PN3211 ultraviolet spectrophotometric (UV) detector (Postnova Analytics, Landsberg) using a deuter- ium lamp set at 254 nm, with a PN3621 Multi Angle Laser Light Scattering Detector/MALLS (Postnova Analytics, Landsberg) and with a DLS detector in line in Zetasizer PN3702 (Malvern Instruments, UK). The acquisition and processing of data were performed using AF2000 Focus Software Version 184.108.40.206. The geometric radius (R g ) of the
Cell viability analysis by Sulforhodamine B (SRB) assay Cells were harvested in log phase, then seeded into 96-well cell culture plates. MCF-7 cells were washed with PBS and then transferred to phenol red-free DMEM containing double char- coal-stripped serum after 48 h, and incubated for a further 48 h. MCF-7/LCC1 and MCF-7/LCC9 cells were maintained in double charcoal-stripped medium for 96 h. Cells were then treated with STAT inhibitors. The STAT1 inhibitor (-)-epigallo- catechin gallate (EGCG) is a major component of green tea and was obtained from Sigma Aldrich . The STAT3 inhibitors Stattic [24,25] and WP1066  were both obtained from Calbiochem. Stattic is a non-peptide small molecule inhibitor reported to inhibit STAT3 dimerization by selectively interacting with the STAT3 SH2 domain . WP1066 has been shown to inhibit STAT3 signal pathway by down-regulating STAT3 targets and activating Bax to inhibit STAT3 nuclear localization . Recent data has suggested that Stattic may also interact with STAT1, so it is feasible that some of its action may be mediated via STAT1 . After incubation for 5 days with inhibitors, cells were fixed using 25% cold trichloroacetic acid (Sigma), and incubated for 1 h at 4 uC. Plates then were washed, air-dried, and stained with sulforhodamine B (Sigma) dye (0.4% solution in 1% acetic acid). After being washed with 1% acetic acid, Tris buffer (10 mM, pH 10.5) was added to each well 1 h prior to the optical density (OD) being read using a Biohit BP800 Microplate reader at 540 nm.
It is possible that, following chemotherapy, recurrent tumors show enhanced malignancy due to an increased percentage ofcancer stem cells. Indeed, Dylla et al. (16) reported that human colorectal cancer stem cells were enriched in colon tumors following chemotherapy and remained capable of rapidly regenerating the tumors from which they originated. Similar results were observed following chemotherapy of thyroid carcinoma (17). One possibility is that cancer stem cells remain at rest in the G0 phase of the cell cycle, and are thus not sensitive to chemotherapy because most drugs selectively kill prolifer- ating cells (18). Conversely, the genomic instability of tumor cells may contribute to the new generation ofcancer stem cells, and this process may be potentiated by exposure to chemotherapeutic agents (19). Cancer stem cells exhibit several characteristics, such as an active DNA repair system and expression of anti-apoptotic factors, which may promote chemotherapeutic resistance. Consequently, com- plete disease remission might only be obtained after total elimination of the cancer stem cell population (19).
Selaginella tamariscina was purchased from herb stores and dried whole plants (100 g) were extracted twice with 500 ml of 50% ethanol in distilled water. The pooled extracts were filtered and concentrated at 70°C using a rotary evaporator under low pressure. The concentrated crude extract was frozen at −80°C for 2-3 days and then it was freeze-dried in a lyophilizer and stored at −20°C. The extraction yield was 2.8% (w/w) and the chemical profile of Selaginella tamariscina extract (STE) was analyzed by using high-pressure liquid chromatograms (HPLC)-mass spectrometer . Briefly, Selaginella tamariscina were analysed by HPLC- mass spectrometer using a HPLC (Hitachi L-6200 with an L-4500 Diode Array detector) with a PE Sciex Qstar Pulsar ESI-TOF mass spectrometer. Samples (10 µl) were injected onto a Merck LiChrospher 100 RP-18 column (4 x 250 mm). The column was equilibrated in 0.05% acetic acid/water (solution A) and elution of the components was achieved by increasing the concentration of solution B (100% acetonitrile) from 0 to 100% in 30 min at a flow rate of 1 ml/min. Absorbance was monitored at 254 nm. The molecular masses of the peaks were determined from electrospray ionisation mass spectra using multiply- charged ion profile . The extract was dissolved in dimethyl sulfoxide (DMSO) (Sigma Co., USA) and was prepared at different concentrations for the subsequent experiments.
Oligos microarray chips ( ,57K genes) were obtained from GE HealthCare (IL) and AppliedMicroarrays (MA) (CodeLink 57K Human Whole Genome). Hybridization was performed with the CodeLink RNA amplification and Labeling kit, utilizing the Cy5 fluorescent dye. Slides were scanned with a microarray scanner (ScanArray 4000XL). Images were generated with ScanArray microarray acquisition software (GSI Lumonics, USA). cRNAs from three experimental setups were used in single experiments with internal spikes as controls. The experimental setups consisted of 10 urinary BC samples of different histologies (T1/2-grade 3, T1-grade 1/2, T3-grade 3) and 5 control samples. The scanned images were further processed with the CodeLink Expression Analysis Software v5.0 from Amersham Biosciences. The experimental setup was analyzed based on the reference design as described previously [9,10,11]. All tumor samples were compared against the mean value of the control samples. Background correction was performed by subtracting the median global background from the median local background from the signal intensity. A threshold of 2 was set as cut-off, meaning that spot intensity for at least one channel should be twice as much as that of the background. Microarray data were normalized by dividing spot intensities by the global median. Normalized data were extracted, pre-processed and sorted with Microsoft ExcelH. Array data are available at the Gene Expression Omnibus (National Center for Biotechnology Information) with accession numbers GSM678186 through GSM678385 (http://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE27448). Furthermore, each gene was tested for its significance in differential expression using a z-test. Genes were considered to be significantly differentially expressed if they obtained a p-value ,0.05. The False Discovery Rate was calculated as described previously [12,13,14]. Genes were further classified using two-way (genes- against samples) average-linkage hierarchical clustering with Euclidian distance using the Genesis 1.7.2 software (Technische Universitaet-Graz, Austria) .
sequences which can flank break points. Repetitive sequences result in ambiguous mapping and PETs with tags mapping to repetitive regions are excluded. The 1 kb libraries were advan- tageous in identifying deletions with span ,5 kb. The larger number of deletions identified by 1 kb libraries is due to the more precise insert size selection and thereby smaller standard deviation of the insert size distribution. This results in a higher sensitivity for the identification of small deletions since deletions are identified by a PET mapping distance which is larger than the general insert size distribution of the respective library. However, 10 kb libraries had a comparable resolution in predicting breakpoint locations to a distance that can be amplified by PCR. This is important to note as large insert libraries are often believed to have a proportionally lower breakpoint resolution. The 20 kb insert size library requires a more stringent cluster count cutoff and might have a slight advantage in discovering inversions and unpaired inversions but displayed a lower sensitivity in identifying small SVs of various categories compared to 10 kb insert size library, whereas the construction of libraries with 20 kb inserts requires more genomic DNA as starting material. The detailed characterizations of SVs by large insert size libraries showed many new sub-types of insertions, which could help in understanding the genesis and effect of insertions in human normal and cancer genomes.
For more than a century, pathological examinations have been the primary and most important tool for the diagnosis of cancerous regions. Cancer classification itself has been established based on the findings of classical staining methods such as HE staining, and such methods will continue to play a leading role incancer diagnosis. However, the limitations of classification based on classical staining findings should be noted. It is often the case that patients with the same pathological diagnosis do not always have the same prognosis. Diagnoses are often made based on morphology. Using conventional pathological techniques, we can only conduct morphological observations, and it is difficult to reveal the details of components in tissue sections. In situ hybridization and/or immunohistochemistry analyses enable the analysis of the distribution of known molecules; however, it has remained impossible to examine the distribution of unknown molecules. For a detailed and accurate diagnosis, it is necessary to obtain information regarding components such as specific proteins and lipids in a sample.
F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressedin breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancercell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues.
nature, depending on parameters which vary between different cell types as is discussed in Ref. 20. Profilin 1 may either sequester G-actin and by that inhibit actin polymerization or promote actin assembly . As tumor suppressor, profilin 1 was reported many times to inhibit cell motility and matrigel invasivness [20,28,35], but exact mechanism has not been discovered yet. We showed that PC-3 cells with inhibited cathepsin X had higher F-actin content (filamentous actin) than cells with active cathepsin X. In literature, for profilin 1-specific siRNA treated MDA-MB-231 cells, reduced F-actin content and reduced F-actin staining near the leading edge were shown , whereas overexpressed or microinjected profilin 1 increased overall F-actin . We may hypothesize that by cleaving C-terminal of profilin 1, cathepsin X regulates actin polymerization and consequently cell migration. The cleavage of profilin 1 may interfere also binding to multidomain proteins like Arp2/3 complex, WASP and VASP which are poly-L-proline rich ligands with binding sites for profilin 1 and actin (in globular and filamentous form). They act as recruiters of the profilin 1/actin complexes to the sites of filamentous actin elongation near the plasma membrane . The cleavage of profilin 1 may also interfere the binding to phosphoinositides, another important molecules regulating cell motility .
Studies show the important roles for some chemokines in hematopoiesis and organ development. Hematopoiesis is an active progression controlled by a variety of cytokines. It is well clear that the chemokine family plays an critical role in this regulatory system. At least 25 chemokines of the CC, CXC and C subgroups have been found to sup- press the in vitro proliferation of myeloid progenitor cells (12, 13). However, the in vivo evidence based on knock- out mice studies provides data of contrasting hematopoi- etic effect for only few of the chemokines and their recep- tors. For example, CCL3 (MIP-1α) arrests cell cycling and reduces the bone marrow progenitor cells number in mice (14). Mice depleted of CCR1, as CCL3 receptor, display enhanced lineage-committed myeloproliferation and leukocyte mobilization into the blood stream (15). CXCL12 (SDF-1) is constitutively expressed by stromal cells in bone marrow, promotes proliferation of B cell progenitors (16), and recruit hematopoietic precursors to the bone marrow in embryogenesis time (17). Mice de- ficient in CXCL12 (SDF-1) or its receptor (CXCR4) die before birth with in B lymphopoiesis and myelopoiesis deficiency and also with an incomplete development of the cardiac septums and cerebellum development, it show the involvement of CXCL12 /CXCR4 pair in a number of vital developmental processes (18-20). The thymus is organ for T cell development and it expresses mRNA for several chemokines with lymphocyte-attracting proper- ties, that well described (21,22). Some chemokines con- trol the development and organization of secondary lym- phoid organs such as peripheral lymph nodes and Peyer’s patches (23,24).
The identification of galectin-7 as a p53-induced gene and its ability to induce apoptosis in many cell types support the hypothesis that galectin-7 has strong antitumor activity. This has been well documented in colon cancer. However, in some cases, such as breast cancer and lymphoma, its high expression level correlates with aggressive subtypes ofcancer, suggesting that galectin-7 may have a dual role incancer progression. In fact, in breast cancer, overexpression of galectin-7 alone is sufficient to promote metastasis to the bone and lung. In the present work, we investigated the expression and function of galectin-7 in melanoma. An analysis of datasets obtained from whole-genome profiling of human melanoma tissues revealed that galectin-7 mRNA was detected in more than 90% of biopsies of patients with nevi while its expression was more rarely found in biopsies collected from patients with malignant melanoma. This frequency, however, was likely due to the presence of normal epidermis tissues in biopsies, as shown our studies at the protein level by immunohistochemical analysis. Using the experimental melanoma B16F1 cell line, we found that melanoma cells can express galectin-7 at the primary tumor site and in lung metastasis. Moreover, we found that overexpression of galectin-7 increased the resistance of melanoma cells to apoptosis while inducing de novo egr-1 expression. Overexpression of galectin-7, however, was insufficient to modulate the growth of tumors induced by the subcutaneous injection of B16F1 cells. It also failed to modulate the dissemination of B16F1 cells to the lung.
Introduction. CD44 has been proposed as a prognostic marker and a stem cell marker but studies in patients with prostate cancer have yielded inconsistent results. Patients and Methods. Patients submitted to radical prostatectomy between 2008 and 2013 at a university hospital were followed with biannual serum PSA tests to determine the biochemical recurrence (BR). Archived paraffin blocks with neoplastic and nonneoplastic tissue were evaluated immunohistochemically for a panCD44 and MYC. Results. Sixty- nine patients completed follow-up and were included. CD44 positivity was observed in inflammatory cells (42%), nonneoplastic epithelium (39.7%), and neoplastic tissue (12.3%). In nonneoplastic tissues staining was observed in basal and luminal cells with the morphology of terminally differentiated cells. In neoplastic tissues, CD44 negativity was correlated with higher Gleason scores (Rho = −0.204; 𝑝 = 0.042) and higher preoperative serum PSA levels when evaluated continuously (𝑝 = 0.029). CD44 expression was not associated with tumor stage ( 𝑝 = 0.668), surgical margin status (𝑝 = 0.471), or BR (𝑝 = 0.346), nor was there any association between CD44 and MYC expression in neoplastic tissue ( 𝑝 = 1.0). Conclusion. In the bulk of cells, the minority ofcancer stem cells would not be detected by immunohistochemistry using panCD44. As a prognostic marker, its expression was weakly correlated with Gleason score and preoperative PSA level, but not with surgical margin status, tumor stage, or BR.
Tissue samples were obtained from 23 unrelated Korean primary gastric cancer patients that underwent surgical resection at Pusan National University Hospital and Pusan National Uni- versity Yangsan Hospital and provided written informed consent. The study was approved by the Pusan National University Hospital-Institutional Review Board (PNUH-IRB) and the Pusan National University Yangsan Hospital-Institutional Review Board (PNUYH-IRB). Total RNA from tissues and cells were extracted using Trizol reagent (Invitrogen) or an RNeasy Mini kit (Qiagen, Valencia, CA, USA), according to the manufacturers' instructions. Quantitative real- time polymerase chain reaction (real-time PCR) was used to check DPY30 mRNA levels. cDNAs were synthesized with MMLV reverse transcriptase (Promega, Madison, WI, USA), dNTP, and oligo-dT primers. The primer sequences used were as follows: DPY30, 5’- AAC GCA GGT TGC AGA AAA TCC T -3’ and 5’- TCT GAT CCA GGT AGG CAC GAG -3’; WDR5, 5’- TGT TAC TGG TGG GAA GTG GA -3 and 5’- CTG TTG GGT GAC AAG CTG TT -3’; RBBP5, 5’- AGT GCA CAC ATC CAT CCA GT -3’ and 5’- TCA CAG TCG CCT GAA AGA AC -3’; ASH2L, 5’- TAC AAG AGC TGC ACG GTT TC -3’ and 5’- CCA GCC CAT GTC ACT CAT AG -3’; GAPDH, 5’- TGG GCC AGG AAA TCA CAT CC -3’ and 5’- CTC AGC CCG AGT GGA AAT GG -3’. Real-time PCR was performed using a LightCycler™ 96 Real-time PCR system (Roche, Nutley, NJ) and FastStart Essential DNA Green Master (Roche), according to the manufacturer's instructions. GAPDH was used as an internal control.
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The macrophage migration inhibitory factor (MIF) has been increasingly implicated in can- cer development and progression by promoting inflammation, angiogenesis, tumor cell sur- vival and immune suppression. MIF is overexpressed in a variety of solid tumor types in part due to its responsiveness to hypoxia inducible factor (HIF) driven transcriptional activation. MIF secretion, however, is a poorly understood process owing to the fact that MIF is a lead- erless polypeptide that follows a non-classical secretory pathway. Better understanding of MIF processing and release could have therapeutic implications. Here, we have discovered that ionizing radiation (IR) and other DNA damaging stresses can induce robust MIF secre- tion in several cancercell lines. MIF secretion by IR appears independent of ABCA1, a cholesterol efflux pump that has been implicated previously in MIF secretion. However, MIF secretion is robustly induced by oxidative stress. Importantly, MIF secretion can be observed both incell culture models as well as in tumors in mice in vivo. Rapid depletion of MIF from tumor cells observed immunohistochemically is coincident with elevated circulat- ing MIF detected in the blood sera of irradiated mice. Given the robust tumor promoting activities of MIF, our results suggest that an innate host response to genotoxic stress may mitigate the beneficial effects ofcancer therapy, and that MIF inhibition may improve thera- peutic responses.
who have never smoked. Among nonsmokers, passive smoking and exposure to other carcino- gens are risk factors for the development of the tumor. In both cases, the hereditary component is determinant, since it confers a greater genetic predisposition to the individuals. Genes such as p53, p14ARF, p16INK4a, RB, FHIT and RASSF1A are related to lung cancer. (6-10) In addition to
After the first tubulin-binding agents showed antitumor effects, many other tubulin-targeting antimitotic drugs were developed, along with related inhibitors directed at other targets such as kinases and mitotic protein complexes, aimed at inhibiting cell proliferation (5). Among the MAPs, the kinesin spindle protein (KSP) Eg5 is a kinesin responsible for the separation of centrosomes and for the bipolar configura- tion of the mitotic spindle. Inhibition of Eg5 causes monopolar spindle formation and cell cycle arrest, an effect demonstrated by monastrol, a small-molecule Eg5 inhibitor (10,11). Other Eg5 inhibitors, such as ispinesib (Cytokinetics), SB743921 (Cytokinetics), AZD4877 (AstraZeneca), Arq621 (ArQule), EMD-534085 (Merck-K GaA), MK-0731 (Merck & Co.), filanesib (Array Biopharma) and litronesib (Kyowa Hakko Kirin/Eli Lilly), have reached clinical trials (12). Centromeric protein E (CENP-E) is another MAP inhibited by antitumor agents. CENP-E is essential for the alignment of chromo- somes during metaphase and passage to anaphase (3). Examples of CENP-E inhibitors are PF-2771 (13), UA62784 (14) and Cmpd-A (15), but only GSK923295 has reached clinical trials (16). Abnormalities in the function and exp- ression of kinesins are important for the development and progression of many human cancers, suggesting that this class of proteins is an interesting target for new anti- cancer therapy strategies (17,18). Since some kinesins are expressed only during mitosis, their inhibition may reduce side effects related to antineoplastic agents targeting inter- phase proteins, such as traditional tubulintargeting antimi- totics (9).